Gliederung

Mutations in the intermediate filament protein desmin cause a distinct class of myofibrillar myopathies that are characterized by deposition of desmin aggregates. To assess the effect of different disease-associated mutations at the molecular level, we applied dual color confocal single-particle fluorescence spectroscopy along with fluorescence microscopy. We studied thede novoaggregation properties of desminin vitroand the aggregation state of desmin-GFP and -mKate2 fusion proteins in homogenates of transfected cells. We detected divergent assembly patterns for three different desmin missense mutations. R350P-desmin showed a strong inhibition of assembly formation that was associated with a reduced level of tetramers and an increase in dimers in native cell extracts. E413K-desmin formed hyperstable tetramers. For R454W-desmin, there were subtle effects on assembly at the dimer and tetramer levels by single-particle spectroscopy that are not detectable by conventional fluorescence microscopy. R350P-desmin efficiently interacts with the wild-type protein resulting in a dominant-negative effect on desmin assembly. To characterize the architecture of pathological aggregates formed by mutant desmin in cellular and molecular models of myofibrillar myopathies and to better understand the regulation of the formation of either physiological assemblies or pathological aggregates by desmin at the molecular level we used a range of confocal fluorescence-based multi-color approaches including dual-color SIFT and single-particle FRET. Taken together, our results provide a molecular basis for a detailed functional classification of mutations in the desmin gene. The findings may also have implications for diagnostic and therapeutic strategies for primary desminopathies based on various molecular events that disrupt physiological filament formation.